Use of Conductance to Detect Bacteriocin Activity

772 Journal of Food Protection, Vol. 53, No. 9, Pages 772-776 [September 1990) Copyright International Association of Milk, Food and Environmental Sanitarians Use of Conductance to Detect Bacteriocin Activity GIORGIO GIRAFFA,* ERASMO NEVIANI, and ADELIA VENERONI lstituto Sperimentale Lattiero Caseario - 20075 Lodi (Italy) (Received for Publication November 6, 1989) ABSTRACT The inhibitory activity of a bacteriocin produced by Lactobacillus delbrueckii subsp. lactis G4 (Bac + ) in milk was investigated by using conductivity measurements. The bacteriocin showed an inhibitory action toward some strains belonging to L. delbrueckii subsp. bulgaricus species. A delay in detection time (5DT) of two milk cultures sensitive to bacteriocin, grown in the presence of preformed bacteriocin, was observed. An inactivation as well as a modified growth rate of the sensitive cultures due to bacteriocin activity might explain the 5DT, as indicated by longer generation time (tg). Cells showed the highest sensitivity to bacteriocin during the log phase of growth that corresponded to the beginning of the acceleration of the conductance curve (DT). Bacteriocims are antimicrobial compounds possessing an inhibitory activity toward other microorganisms. Criteria generally adopted to define bacteriocins come from colicins and other well-studied bacteriocins. They mainly show a narrow spectrum of activity, a proteinaceous nature, and a bactericidal mode of action (6,13). The literature reports several studies on bacteriocins, describing the presence of a wide class of heterogeneous molecules possessing different spectra of inhibition, mechanisms of action, and chemical nature (6,13). Among the lactic acid bacteria, bacteriocins have been extensively studied in streptococci. In this regard, two groups of substances have been described: one exhibiting a more typical narrow range of antibacterial activity and a second group effective against a wide number of gram-positive species (5,13,15). Rod-shaped lactic acid bacteria have received more limited attention. Literature is available both on bacteriocin expression and production; studies on biochemical properties and genetics are still accumulating (13). Bacteriocins in Lactobacillus genus have been detected in Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus bulgaricus, and Lactobacillus plantarum (2,12,13). Many workers clearly showed that bacteriocin production might be considered as a positive factor in preventing the growth of pathogenic and spoilage bacteria in different foods (4,6,13). From a technological standpoint, there has been little comment on strain domination caused by the bacteriocin presence. Because of the narrow spectrum of activity exhibited by many bacteriocins, their production could cause some imbalance in mixed strain cultures of dairy lactic acid bacteria. Previous studies demonstrated strain competition occurring by bacteriocin production in a mixed strain culture of L. acidophilus grown in MRS broth at ph 6 (8). Also, few attempts have been made to study the mechanism of inhibition of bacteriocin producing strains in milk. This might be useful to better understand how inhibition of sensitive cells occurs in practical cheesemaking conditions. In a previous study (10), the production and the mechanism of inhibition of Lactobacillus delbrueckii subsp. lactis bacteriocins in MRS broth medium were observed. In the present work, we describe the consequences related to bacteriocin production by mixed strain cultures grown in milk, using conductivity measurements. MATERIALS AND METHODS Strains, media, and growth conditions L. delbrueckii subsp. lactis G4 as the bacteriocin producing strain and Lactobacillus delbrueckii subsp. bulgaricus 116 and S54 as the indicator strains were obtained from the stock collection of our Institute. They were maintained as frozen stocks at -20 C in litmus milk. In preparation for experiments, they were cultured in MRS broth (7) at 42 C for 15-18 h. Cell-free bacteriocin supernatants Cell-free bacteriocin supernatants were obtained after centrifugation (7000 rpm, 5 min, 4 C) followed by neutralization with 5N NaOH and filter sterilization (0 0,22 urn, Nuclepore) of an overnight culture of the producer strain. These nonpurified preparations were stable at 4 C for 1-2 d. They were treated at 100 C for 10 min (10) to obtain heat inactivated cell-free bacteriocin supernatants. Bacteriocin detection and quantitative determination L. delbrueckii subsp. lactis G4 was checked for bacteriocin production as described previously (10). MRS agar plates (10 ml, % agar) were overlaid with MRS soft agar (5 ml, 0.75% agar) containing 10 5-10 6 CFU/ml of an overnight culture of L. delbrueckii subsp. bulgaricus 116 as the indicator strain. After *Corresponding author: 1st. Sper. Latt. Cas. - Via Lombardo, 11-20075 diffusion of 10 u.1 of a cell-free bacteriocin supernatant, the Lodi (Italy). plates were incubated at 42 C for 24 h under anaerobic condi-

USE OF CONDUCTANCE TO DETECT BACTERIOCINS 773 tions (Gas Pak System, BBL Lab, Cockeysville, MD). Quantitative determination of the bacteriocin was obtained by the method of Joerger and Klaenhammer (12) and expressed in activity units (AU) /ml. Isolation of nonproducing bacteriocin (Bac-) variants L. delbrueckii subsp. lactis G4 was examined for the occurrence of Bac- variants after repeated transfers in MRS broth and plating on MRS agar. After incubation at 42 C for 15 h, the plates were overlaid with MRS soft agar containing L. delbrueckii subsp. bulgaricus 116 as the indicator strain and incubated for a further 15 h. Variants showing Bac-phenotype were detected for the absence of the zones of inhibition surrounding the colonies. They were isolated from the plates and streaked onto MRS basal plates (MRS without beef extract and glucose) containing % of different carbohydrates for purification. Carbohydrates utilized were sucrose, maltose, and trehalose. Controls included L. delbrueckii subsp. lactis G4 and L. delbrueckii subsp. bulgaricus 116 streaked onto MRS basal agar plates plus tested carbohydrates. Resistance of the Bac- derivatives to bacteriocin was also checked by the plate diffusion method, as described in a previous section, using Bac- variants as the indicator strains. Microbiological growth monitoring Bacterial growth was evaluated by conductance measurements using a Malthus Instrument Growth Analyzer (3,11,17). This system detects changes in conductance caused by the bacterial metabolism in the growth medium. Changes are expressed in Microsiemens (p.s); they are shown graphically as conductance curves. Together with these curves, two parameters can be considered to study the microbial metabolism: a) Conductance detection time (DT), expressed in hours. It is defined as that point where the baseline ends and the region of acceleration of the curve begins; b) Generation time (tg). Correlation between the conductance curve and the growth phase of L. delbrueckii subsp. bulgaricus 116 indicator strain Skimmed milk was dispensed in 9 ml amounts in 10 ml capacity growth cells (Malthus Instruments) and autoclaved at 115 C for 15 min. Each tube was then inoculated with L. delbrueckii subsp. bulgaricus 116 as indicator strain from an overnight culture to reach about 10 5, 10 4, 10 3, 10 2, and 10 CFU/ml. The inoculated series of tubes were incubated at 42 C for 24 h to monitor the conductance curve of the sensitive strain. At different intervals, samples from the culture containing 10 5 CFU/ml as initial population were taken aseptically from the growth analyzer for the enumeration of the viable cells in MRS agar. The plates were incubated at 42 C for 24 h under anaerobic conditions. Inhibition mechanism The loss of viability of the sensitive cells caused by the bacteriocin was determined by two methods. The first consisted of the addition of increasing amounts of bacteriocin in sterile skimmed milk cultures containing about 10 5, 10 4, 10\ 10 2, and 10 CFU/ml of L. delbrueckii subsp. bulgaricus S54 and 116 sensitive strains. The titers of the added bacteriocin were calculated as previously described and expressed in AU/ml. Samples with and without bacteriocin were placed into the growth analyzer to obtain the DT, tg, and conductance curve of each culture after incubation at 42 C for 24 h. The relationship between DT and CFU/ml of different dilutions was used to obtain a calibration curve. The slope of the line is strictly related to the tgs of the cultures that were calculated using the following equation: tg = 0.15 x 8 100 DT (9), where 8 I00 DT is the delay of detection time (in h) evaluated on the calibration line for a 100-fold dilution. To appreciate the effect of the bacteriocin on the metabolism of the sensitive cells, changes in tg and in DT (8DT) between the cultures with and without bacteriocin were considered. Differences of slope and final conductance values of the conductance curve were also evaluated. The second method consisted of comparing the relationship between the growth phase of sensitive cells and the level of inhibitory effect caused by a given amount of bacteriocin. Skimmed milk sterilized and dispensed as above was inoculated with 10 5 CFU/ml of an overnight culture of the 116 indicator strain and incubated at 42 C into the growth analyzer. At different times, corresponding to different growth phases, samples were taken from the tubes and diluted to reach the same cell concentration. Dilution factors were calculated using the growth curve of a culture containing 10 5 CFU/ml as initial population obtained previously. Diluted samples were used to inoculate new tubes (subcultures) containing sterile skimmed milk plus 6400 AU/ml of a cell-free bacteriocin supernatant, to obtain an initial cell concentration of approximately 10 4 CFU/ml. Control tubes were similarly inoculated. They included sterile skimmed milk + MRS broth; sterile skimmed milk + heat inactivated cell-free bacteriocin supernatant; and sterile skim milk + cell-free MRS supernatant from a Bac- isolate of L. delbrueckii subsp. lactis G4 (positive control). The ratio between skimmed milk and the different MRS broth samples was 50:50 (v:v). 8 t DT was defined as the delay of DT between subcultures containing bacteriocin and heat inactivated bacteriocin and 8 2 DT between subcultures containing bacteriocin and cell-free preparations from a Bac- isolate. The influence of the bacterial growth phase on the effectiveness of the bacteriocin action was assessed evaluating 8jDTs, 8 2 DTs, and the conductance curves of the cultures. RESULTS Characterization of a Bac- variant The Bac- variants were classified as L. delbrueckii subsp. lactis according to characteristics of this species described in the 8th edition of Bergey's (18). Inhibition studies The comparison between the growth curve and the conductance curve of L. delbrueckii subsp. bulgaricus 116 with an initial cell concentration of 10 5 CFU/ml (Fig. 1) showed that the DT, that represents the point of acceleration of the latter curve, occurred during the log phase in the growth curve. The number of viable cells corresponding to DT was 2xl0 7 CFU/ml and was the same for all the other cultures with different initial cell numbers. Increasing the amount of bacteriocin (from 140 to 4300 AU/ml) caused a concomitant increase of SDT of both 116 and S54 cultures. The lower the initial cell concentration of organisms the more affected they are by bacteriocin (Table 1). A linear effect of bacteriocin concentration on SDT was observed (Fig. 2) for both strains (S54 and 116). The behavior of SDT permitted us to observe that the sensitivity to bacteriocin was similar for both strains (Table 1 and Fig. 2). The increase of tg caused by bacteriocin action con-

774 GIRAFFA, NEVIANI AND VENERONI I? 8 6 0-25. «CFU 0,uS / " / / -1 IL 0 3 6 9 1224 HOURS Figure 1. Correlation between the conductance and the growth curves of Lactobacillus delbrueckii subsp. bulgaricus 116 cultivated in milk. TABLE 1. Killing activity of increasing amounts of bacteriocin (expressed in activity units, AU/ml) against milk cultures of Lactobacillus delbrueckii subsp. bulgaricus 116 and S54. 10 to 10 5 represent the initial inoculum rate of the different cultures. 5DT represent the delay of detection time among the cultures with and without the bacteriocin. Strains AU/ml of bacteriocin CFU/ml 200 700 1400 2800 4200 8DT 116 S54 10 1 10 2 10 3 10 4 10 5 10 1 10 2 10 3 10 4 10 5 0.4 0.3 0.1 0.8 0.7 0.7 0.6 1.8 1.0 2.2 3.5 3.3 3.4 1.8 2.2 6.8 5.0 4.0 7.0 5.6 4.1 4.2 8.2 7.4 6.6 6.2 5.2 firmed the similar sensitivity to bacteriocin of the two strains (Fig. 3). In fact, the changes in tg with increased amounts of bacteriocin are quite similar for both organisms. The difference in the initial tg is a strain-dependent characteristic. When cells of L. delbrueckii subsp. bulgaricus 116 were subcultured in milk containing preformed bacteriocin, they showed longer detection time than cells grown both in milk + heat inactivated bacteriocin (SjDT) and in milk + cell-free preparations from a Bac- isolate (8 2 DT) (Table 2). Bactericidal activity of the inhibitor against L. * WOO 30W AU/ml Figure 2. Behavior of the delay in detection times (8DT) of Lactobacillus delbrueckii subsp. bulgaricus 116 and S54 milk cultures with initial cell concentration of 10 2 CFU/ml caused by increasing amounts of bacteriocin (expressed in activity units, AU/ml). minj e ^ w> ^ 80 60 40 20 0 0 1000 3000 AU/ml Figure 3. Behavior of the generation time (tg) of Lactobacillus delbrueckii subsp. bulgaricus 116 and S54 caused by increasing amounts of bacteriocin (expressed in activity units, AU/ml).

TABLE 2. 8 DT (expressed in h) of subcultures of Lactobacillus delbrueckii subsp. bulgaricus 116 obtained after inoculation into milk of cells from a culture at different times of incubation, corresponding to different phases of growth (see text). 8 tdt = 8 DT for subcultures containing bacteriocin, and heat inactivated bacteriocin. 8 2DT = 8 DT for subcultures containing bacteriocin and cell free preparations from a Bac- isolate. Growth phase of the culture Lag Time of incubation (h) 0 1.6 USE OF CONDUCTANCE TO DETECT BACTERIOCINS 775 8,DT +0.9 + 1.1 5 2 DT +0.4 + 1.6 Log Stationary Mortality 4.0 5.6 (DT) 6.2 7.5 8.9 9.6 24.0 +0.8 + + 1.9 + 1.7 - + + 1.4 + + + 1.4 + 1.8-0.1 - + delbrueckii subsp. bulgaricus 116 varied with different growth phases, as shown by 5jDT and 5 2 DT for both types of milk subcultures (Table 2). Results indicated that cells were more sensitive to the bacteriocin after the DT of the culture, corresponding to the log phase of growth, especially between 5.6 and 7.5 h of incubation. Cells appeared more resistant during the stationary phase of growth (Table 2). Comparable results were observed with the conductance curves. Examples are shown with the conductance curves of L. delbrueckii subsp. bulgaricus 116 subcultures obtained from a culture during the second part of the log phase of growth (Fig. 4). The graphs show consistent differences of slope and final conductance values between the subculture grown in the presence of 3200 AU/ml of bacteriocin (Fig. 4, line 1) and its control grown in the presence of heat inactivated bacteriocin (Fig 4, line 2). DISCUSSION As stated previously (70), L. delbrueckii subsp. lactis G4 was able to produce a bacteriocin possessing a narrow spectrum of activity. The heat sensitivity of the inhibitor allowed bacteriocin-free preparations to be obtained from MRS broth cultures. The slight bacteriocin sensitivity of a Bac isolate could possibly involve a common genetic site of origin for bacteriocin production and resistance phenotypes. These results strongly suggest an inhibitory activity of the bacteriocin in milk. DT is defined as the time interval between the start of conductance monitoring and the beginning of the acceleration phase of the signal. This acceleration is related to the production of low molecular weight charge metabolites above a certain threshold level. It usually corresponds to a defined number of cells ranging from 10 6 to 10 7 CFU/ml, depending on the species, media, 0 8 12 16 20 HOURS Figure 4. Correlation between the growth phase of Lactobacillus delbrueckii subsp. bulgaricus 116 and its sensitivity to the bacteriocin. Key: 1 represents a subculture in milk + 3200 AUI ml of bacteriocin, 2 represents a subculture in milk + heat inactivated bacteriocin preparation. For all the subcultures, cells from a culture during the second part of the log phase of growth were used. and growth conditions. For the bacteriocin sensitive strain L. delbrueckii subsp. bulgaricus 116, this number was 2xl0 7 CFU/ml for all the initial cell concentrations between 10 CFU/ml and 10 6 CFU/ml. Preformed bacteriocin added to milk cultures of bacteriocin sensitive strains S54 and 116 caused a delay of DT (5DT) that might be explained by a killing activity affecting the number of initial viable cells. This observation is strongly supported by a previous study on bacteriocins from L. delbrueckii subsp. lactis (10). Also, a modified growth rate of the sensitive strains due to bacteriocin activity might explain the 5DT, as indicated by a longer tg. As stated elsewhere, the behavior of 8DT and tg showed a similar sensitivity to bacteriocin for the strains 116 and S54. This could indicate a comparable killing rate of the bacteriocin on the two strains. A linear relationship between 5DT and bacteriocin concentration was obtained (10 2 CFU/ml), suggesting the potential utilization of conductance to assess levels of bacteriocin. Previous studies have shown a relationship between the growth phase and bacteriocin sensitivity (2,8,13). Inhibition tests clearly demonstrated a growth phase involvement of sensitive cells on bacteriocin activity. Cells were highly sensitive during the log phase of growth corresponding to the acceleration of the conductance curve (DT). The technological implication is that bacteriocin could cause a serious failure in acid development.

776 GIRAFFA, NEVIANI AND VENERONI Bacteriocin production among lactic acid bacteria is well documented and studies on the mechanism of action and kinetics of synthesis are accumulating (1,2,4,6,8,10, 13,14,20). Few reports are available on bacteriocin production and activity in milk and cheeses. Moreover, the narrow spectrum of activity of many bacteriocins may produce a strain imbalance in mixed strain starters. This study showed the ability of a bacteriocin producing strain of L. delbrueckii subsp. lactis to inhibit the growth of some sensitive strains belonging to L. delbrueckii subsp. bulgaricus species in milk. Preformed bacteriocin is able to interfere with growth and metabolism of sensitive strains. Bacteriocin from L. delbrueckii subsp. lactis G4 is produced during the log phase of growth (10). This observation together with the high sensitivity of L. delbrueckii subsp. bulgaricus 116 during the log phase of growth, shown in the present study, might lead to a complete strain domination of the former in cultures composed of the two strains. Thus, a practical and rapid method to control bacteriocin presence in a starter and in milk would be desirable. Conductance microbiology is widely used to detect bacteriophage problems in cheesemaking and antibiotics in milk (16,19). Results suggested that conductance methods could be used to screen many strains for production of bacteriocins after cross-reaction of their neutralized and filter sterilized broth cultures against single strain milk cultures of each strain. Moreover, the spectrum of activity of bacteriocins against closely and not closely related species might be evaluated. Compared to the usual agar spot test and well diffusion assay used to screen for antagonistic activities (12,14,15), conductance methodology could offer two main advantages: firstly, many strains might be screened at the same time and inhibition be evaluated more rapidly. Secondly, an inhibitory effect would be monitored by some parameters (as DT or conductance curve) showing a dynamic metabolic relationship between bacteriocin and sensitive cells, not adequately expressed by the evaluation of zones of inhibition surrounding a bacteriocin containing spot. ACKNOWLEDGMENTS The authors wish to thank Dr. D. Taylor of Malthus Instruments for help in the English writing of this publication and Dr. S. Carini for helpful suggestions. REFERENCES 1. Abdel-Bar, N N. D. Harris, and R. L. Rill. 1987. Purification and properties of an antimicrobial substance produced by Lactobacillus bulgaricus. J. Food Sci. 52:411-415. 2. Andersson, R. E., M. A. Daeschel, and H. M. Hassan. 1988. 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